Modulation of Escherichia coli adenylyl cyclase activity by catalytic-site mutants of protein IIA(Glc) of the phosphoenolpyruvate:sugar phosphotransferase system.
It is demonstrated here that in Escherichia coli, the phosphorylated form of the glucose-specific phosphocarrier protein IIA(Glc) of the phosphoenolpyruvate:sugar phosphotransferase system is an activator of adenylyl cyclase and that unphosphorylated IIA(Glc) has no effect on the basal activity of adenylyl cyclase. To elucidate the specific role of IIA(Glc) phosphorylation in the regulation of adenylyl cyclase activity, both the phosphorylatable histidine (H90) and the interactive histidine (H75) of IIA(Glc) were mutated by site-directed mutagenesis to glutamine and glutamate. Wild-type IIA(Glc) and the H75Q mutant, in which the histidine in position 75 has been replaced by glutamine, were phosphorylated by the phosphohistidine-containing phosphocarrier protein (HPr-P) and were equally potent activators of adenylyl cyclase. Neither the H90Q nor the H90E mutant of IIA(Glc) was phosphorylated by HPr-P, and both failed to activate adenylyl cyclase. Furthermore, replacement of H75 by glutamate inhibited the appearance of a steady-state level of phosphorylation of H90 of this mutant protein by HPr-P, yet the H75E mutant of IIA(Glc) was a partial activator of adenylyl cyclase. The H75E H90A double mutant, which cannot be phosphorylated, did not activate adenylyl cyclase. This suggests that the H75E mutant was transiently phosphorylated by HPr-P but the steady-state level of the phosphorylated form of the mutant protein was decreased due to the repulsive forces of the negatively charged glutamate at position 75 in the catalytic pocket. These results are discussed in the context of the proximity of H75 and H90 in the IIA(Glc) structure and the disposition of the negative charge in the modeled glutamate mutants.
Reddy
P
P
Biotechnology Division, Chemical Science and Technology Laboratory, National Institute of Standards and Technology, Gaithersburg, Maryland 20899-0001, USA. prasad@enh.nist.gov
Kamireddi
M
M
D000262:Adenylyl Cyclases / Q000378:metabolism*; D002384:Catalysis; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D006639:Histidine / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011487:Protein Conformation
D029968:Escherichia coli Proteins; C032242:crr protein, E coli; D006639:Histidine; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; D000262:Adenylyl Cyclases